Introduction Motor neuron loss is the primary pathologic feature of amyotrophic lateral sclerosis (ALS). An estimate of the number of surviving motor units (MUs) represents a direct measure of the disease state in ALS. The objective of the study is to compare MU number estimation (MUNE) using the multipoint stimulation method (MPS) and compound muscle action potentials (CMAP) amplitude in patients with ALS. Methods Twenty-eight patients with ALS with a disease duration of 3–48 months were studied. Nerve conduction study of the median, ulnar, tibial, common peroneal, and sural nerves were done. Besides, electromyography (EMG) of cranio-bulbar, cervical, thoracic and lumbosacral muscles, and MPS-MUNE. Results MUNE is decreased in patients with ALS. MUNE was positively correlated with CMAP amplitude, medical research council (MRC) scale, and ALS functional rating scale (ALS-FRS). On the contrary, MUNE was negatively correlated with MUAP duration. Case detection by the MUNE methods was high as compared to that of CMAP amplitude. Conclusions MUNE is highly specific and more sensitive than CMAP amplitude in detecting the neurophysiologic abnormalities in patients with ALS. Case detection by MUNE is three times more than CMAP amplitudes. The rate of decline of motor units using the MPS-MUNE is more sensitive than the MRC score and ALSFRS-R when expressed as the percentage change from baseline.